Characterization of Novel Virulent Broad-Host-Range Phages of Xylella fastidiosa and Xanthomonas

Abstract

The xylem-limited bacterium Xylella fastidiosa is the causal agent of several plant diseases, most notably Pierce's disease of grape and citrus variegated chlorosis. We report the isolation and characterization of the first virulent phages for X. fastidiosa, siphophages Sano and Salvo and podophages Prado and Paz, with a host range that includes Xanthomonas spp. Phages propagated on homologous hosts had observed adsorption rate constants of ϳ4 ؋ 10 ؊12 ml cell ؊1 min ؊1 for X. fastidiosa strain Temecula 1 and ϳ5 ؋ 10 ؊10 to 7 ؋ 10 ؊10 ml cell ؊1 min ؊1 for Xanthomonas strain EC-12. Sano and Salvo exhibit >80% nucleotide identity to each other in aligned regions and are syntenic to phage BcepNazgul. We propose that phage BcepNazgul is the founding member of a novel phage type, to which Sano and Salvo belong. The lysis genes of the Nazgul-like phage type include a gene that encodes an outer membrane lipoprotein endolysin and also spanin gene families that provide insight into the evolution of the lysis pathway for phages of Gram-negative hosts. Prado and Paz, although exhibiting no significant DNA homology to each other, are new members of the phiKMV-like phage type, based on the position of the single-subunit RNA polymerase gene. The four phages are type IV pilus dependent for infection of both X. fastidiosa and Xanthomonas. The phages may be useful as agents for an effective and environmentally responsible strategy for the control of diseases caused by X. fastidiosa.T he plant-pathogenic bacterium Xylella fastidiosa is the causative agent of a number of economically important diseases, including Pierce's disease of grape, phony peach disease, periwinkle wilt, citrus variegated chlorosis, almond leaf scorch, oleander leaf scorch, and coffee leaf scorch (1). Existing disease control methods are often only partially successful and, in the case of systemic insecticides used to control vector populations, may be potentially harmful to the environment (2, 3). Recently, there has been renewed interest in the application of bacteriophages (phage) as an environmentally acceptable mode for the control of bacterial plant disease (4).To this end, our group isolated and characterized the first temperate (lysogenic) phage of X. fastidiosa, Xfas53 (5). However, temperate phage should not be used as biocontrol agents because of their capacity for lysogenic conversion, superinfection immunity, and risk of generalized transduction. Instead, virulent (lytic) phages are needed for an effective and sustainable phage-based control strategy (6). Here we report the isolation and characterization of the first virulent siphophages and podophages for X. fastidiosa that are members of the Nazgul-like phage and the phiKMV-like phage types, respectively. MATERIALS AND METHODSBacterial strains and culture conditions. Bacterial strains and plasmids used in this study are listed in Table 1. X. fastidiosa strains were cultured at 28°C in PW-M broth (PW-MB) (7) or on PW-M agar (PW-MA) plates (PW-MB with 20 g/liter plant cell culture-tested agar [...

Comparative analysis of two bacteriophages of Xanthomonas arboricola pv. juglandis

“…15 terminal repeat at both of its ends, which was also characteristic for the phiKMV-like phages Prado and Paz (Ahern et al, 2014).…”

“…A single unit DNA-dependent RNA polymerase (RNAP) gene was found at the end of the Class II gene cluster. The Xaj24 phage was most similar to the Prado phage (accession number: KF626667); (Ahern et al, 2014), as the two phages shared 67.8 % identity at nucleotide level. The Xaj24 genome contained a 482 bp direct…”

“…All proteins encoded by DNA metabolism genes of Xaj24 were closely related to the appropriate Prado phage proteins at protein level (E-values between 0 -5E-85). Due to the high homology with Prado phage proteins it was not surprising that Xaj24 DNA metabolism proteins resembled their counterpart proteins encoded in other, nonphiKMV-like Xanthomonas phage (CP1, Xop411, Xp10, OP1, phiL7) genomes (Ahern et al, 2014).…”

Complete Genome Sequence of <b><i>Proteus mirabilis</i></b> Phage pPM_01 Isolated from Raw Sewage

“…Open reading frames (ORF) were automatically identified employing Rapid Annotation using Subsystem Technology (RAST) (http://rast.nmpdr.org/) [41] and Geneious software (Biomatters Limited). Additional protein homology searches of nucleotide sequences and manual annotation of putative translated ORF products were carried out using blastx at NCBI (http://blast.ncbi.nlm.nih.gov) against a nonredundant protein sequence database (E value cut-off <10 -4 ) [3] to compare the homologies with known protein sequences. Finally, the genome annotation was mapped using Geneious software.…”

“…Phages are environmentally ubiquitous, with an estimated 10 31 particles on earth [1,2]. Metagenomic analyses have revealed that they are immensely diverse and present in almost all niches where bacterial cells are present [3,4]. Phages have the capability to kill specific bacteria during exponential bacterial growth.…”